Functional characterization of the transcription programs underlying phase transition in the BSL3 pathogen Coccidioides immitis

Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.球孢子菌病（Coccidioidomycosis，又称谷热Valley Fever）是一种新兴的地方性真菌感染性疾病，其发病率逐年攀升，地理分布范围持续扩张。该病由球孢子菌属（Coccidiodes）引发，该菌属于温度依赖性双态真菌（thermally dimorphic fungi）：在土壤中以菌丝体（mycelia）形式生长，而在宿主体内的肺部环境中则会转化为具有致病性的球形体（spherules）。目前学界对这一形态转化背后的调控机制尚未阐明。本研究借助带帽小RNA测序（capped small RNA-seq, csRNA-seq）——该技术可识别主动转录起始的稳定与不稳定转录本，从而以极高灵敏度精准检测基因调控的急性变化——报道了这一高致病性真菌相转化过程中的基因组架构组织特征与关键序列特征变化。球形体转化过程伴随大规模转录重编程、转录本异构体的功能改变，以及非编码RNA（non-coding RNAs, ncRNAs）启动子远端转录水平的显著上调。对球形体激活型调控元件的分析显示，存在一个预测可招募WOPR家族转录因子（WOPR family transcription factor）的基序，而该类转录因子在其他真菌中已被证实为毒力调控因子。本研究鉴定出CIMG_02671为粗球孢子菌（Coccidioides immitis, C. immitis）的WOPR同源蛋白，并证实其可通过依赖WOPR基序的方式激活转录，提示其为致病性相转化的关键调控因子。综上，本研究同时凸显了带帽小RNA测序作为一种高效工具，可用于鉴定调控致病过程的转录机制。